Method for producing inosinic acid and adenylic acid by fermentation



r l I United States Patent METHOD FOR PRODUCING INOSINIC ACID ADENYLIC AQID BY FERMENTATION Shukuo Kinoshita, Shibuya-lru, Tokyo, Shigeo Abe, Suginarni-ku, Tokyo, Kiyoshi Udagawa, Tsururni-ku, Yokohama, Talreshi Saito, Sagamihara-shi, and Kenichiro Talrayama, Setagaya-kn, Tokyo, Japan, assignors to Kyowa Hakko Kogyo Co., Ltd, Tokyo, Japan, a corporation of Japan a No DrawingJjFilcd Mar. 9', 1962, Ser. No. 178,580

Claims priority, application Japan Mar. 11, 1961' Claims, (l. 19528) The present invention relates to a method for producing inosinic acid and adenylic acid by fermentation. According to thisinvention inosinic and adenylic acid are produced by culturing biochemical mutant strains of microorganisms in culturing media containing (a) saccharides or other carbon sources, (b) nitrogen sources, (c) inorganic substances, (d) organic substances and (e) other nutrients necessary for the microorganisms, whereby, g the 5-inosim'c acid (inosine-5'-phosphate), 5- adenylic acid (adenosine-5-phosphate) and 3'-adenylic acid (aderrosine-P:-phosphate) are directly produced and accumulated either singly or in combination in the media.

In further accord with this invention, the acids produced are isolated and separately recovered.

An object of the invention is to provide a novel method for producing inosinic acid andadenylic acid by fermentation. Other objects will be apparent as the description proceeds.

Nucleic acid comprises a chain of repeatiug units, each of which is built up trom a sugar, a purine or pyrimidine base, and phosphoric acid. A'given nucleic acid contains either (but notboth) D-ribose or 2-desoxy-D-ribose as the sugar. Ribonucleic acid (RNA) is a nucleic acid containing rib-ose. Desoxyribonucleic acid (DNA) is a nucleic acid containing Z-desoxy-D-nibose. Genetic control of cellular reproduction is imputed to proteins containing DNA; Inosinic and adenylic acid are building I blocks in the structures of RNA and DNA and therefore can be used as intermediates in nucleic 'acid synthesis. In addition, 3-adenylicacid (yeast adenylic acid) o-roarn gives a quantitative yield of furfural when distilled with 20% 'HCl for three hours.

is readily deaminated by nitrous acid with the formation 1 of inosinic acid. 1; The muscle tissue preparation may be used for the treatment of bursitis, tendinitis, phlebothrombosis and pruritis. (Burger, Alfred, Medicinal Chem- ,istryj" lnterjscience Ifublishers, Inc, New York, second edition, 1960, pager 533; The Merck Indexof Chemicals 3,i52,%b Patented ot. 13, 1964 "Ice hydnolyzes (on boiling with acid) to one mole of each of H PO D-ribose and hypoxanthine.

Each of the above-identified acids, 3'- and 5'-adeny1ic acid and inosinic acid, may be used to enhance the flavor of food, to impart a good taste to food, as a condiment.

, i The microorganisms within the contemplation of this invention are biochemical mutant strains requiring at least one of the substances:

Guanine Thymine 'Hypoxanthine Xanthine for their growth. Microorganisms having this genetic characteristic accumulate inosinic acid and adenylic acid in their culture broths or in their cell bodies. The fact that a microorganism may require one or more additional substances for growth does not adversely affect the property of acid production according to the present invention X-ray, orby treatment with chemical agents of diverse microorganisms which ordinarily do not require guanine, Xanthine, hypoxanthine or thymine for growth. Among microorganisms which can be appropriatelymodified to come within the contemplationof this invention are those of the genera:

Micrococcus, e.g., Micrococcus glutamicus Bacillus, e.g., Bacillus subtilis Escherichia, e.g., Escherichia coli Brevibacterium, e.g., Brevibacterium ammoniagenes Candida, e.g.,'Cana'ida utilis Penicillium, e.g., Penicillz'um ch'rysogenum Aspergillus, e.g., Aspergillus niger Streptomyces, e.g.,'Streptomyces griseus Cal Neurospora, e.g., N eurospora crassa Although the exemplified species, include mutants which do not accumulate practical quantities of 'inosinic and adenylic acids, those mutants which require at least one of the" substances, guaninejxanthi'ne, hypoxanthine 'and thymine, for growth accumulate (upon fermentation) inosinic acid and/ or adenylic acid. t

Up to date, however, exact mechanisms for the accumulations of adenosine-3-phosphate, inosinic acid, or adenosine-5'-phosphate are not yet known, g Y

Of the exemplified biochemical mutants, Micrococcus glutamicus mutants requiring guanine, Xanthine, hypoxanthine, or thymine (or a combination of these substances) can accumulate concurrently a remarkable amount of L- glutamic acid. X 1 1 c Any synthetic ornatural culture mediummay be employed according to the present invention as long as it con-v tains a carbon source, e.g.,'a saccharide such as glucose;

I a nitrogen source, e.g., an organic nitrogen source such as NZ-amine or an inorganic nitrogen source such as ammonium sulfate; inorganic substance, such as KH PO K HPQ; and MgSO -7H O; organicsubstance, such as guanine, xanthine, hypoxanthine or thymine; and. other nutrients, such as biotin. The carbon and nitrogen sources may be any of the known sources which are assimilable by the employed strain.

Carbon sources include various carbohydrates, such as glucose, glycerol, fructose, sucrose, maltose, mannitol, xylose, galactose, lactose, ribose, starch and its hydrolysates, molasses, etc. source is ordinarily from 1 to-20% byweight- (calculated as glucose) based on the volume of the medium. Also, various organic acids, such as gluconic acid, pyruvic acid, fumaric acid, lactic acid, acetic acid, formic acid; various amino-acids, such as glycine, glutamic acid, alanine; and other organic substances, such as glutamine, may be employed;

' Nitrogen sources include ammonia; various inorganic ammonium salts, such as ammonium chloride, ammonium phosphate, ammonium sulfate, amonium nitrate, ammonium carbonate, ammonium acetate; various nitrogenous organic substances, such as urea, peptone, NZ- amine, meat extract, yeast extract, corn steep liquor, casein hydrolysate, fish meal and its digested product, defattedsoy bean and its digested product, chrysalis hydrolysate; and amino acids, such as glycine, glutamic acid, and alanine.

When a medium containing guanine, xanthine, hypoxanthine and/or thymine is employed, guanine, xanthine,

The concentration of the carbon I hypoxanthine, and/ or thymine (including derivatives and I RNA, DNA, etc.), or a material containing these substances, should be added to the medium so that the amount .ofeach substance required for the mutant strain employed is 2 to 90 ,ug/ml. Thus, the total amount in the medium of each substance (as guanine, xanthine, hypoxanthinc, or thymine) required for the growth of the mutant strain employed is desirably such that the effective amount provides a concentration of from 2 to 90 tog/ml. If the amount is in excess, the accumulation of inosinic acid and/or adenylic acid is markedly reduced, though the growth of the microorganism is accelerated. Thus an important condition of the inventive fermentation (to obtain optimum yields of inosinic and/ or adenylic acids) is that the concentration in the medium of said substance(s) required for growth of the microorganism employed (guanine, xanthine, hypoxanthine and/ or thymine) is suboptimum for the growth of said microorganism.

The inorganic substances employed include dipotassiurn hydrogen-phosphate, potassium dihydrogen-phosphate, magnesium sulfate, calcium carbonate, sodium borate, copper sulfate, iron sulfate, manganese chloride, ammonium molybdate and zinc sulfate.

When a mutant strainhaving concurrently a further nutritional requirement, i.e., in addition to guanine, Xanthine, liypoxanthine and/ or thymine, is cultured, the substance which fulfils the further requirement for growth is added to the medium.

The fermentation, according to the present invention, is efiectedunder aerobic conditions, such as by shaking culture or submerged culture withlaeration-agitation. The culturing temperature. is preferably'from 20 to' 40 C. The control of pH of the medium while culturingthe microorganism is exceedingly important. The pH of the medium tends to vary upon commencement of the fermentati'on. It is desirable to adjust and maintain the pH at 6.0 to 7.4 (during cultivation) with a suitable neutralizing agent in order to obtain a higher yield.

As the neutralizing agents, aqueous ammonia, sodium hydroxide, ammonium carbonate, calcium carbonate, etc., may be used. In some cases in which the strain'used has v 4 days cultivation of fungi and Streptomyces may be neces sary before a substantial amount of inosinic acid and/or adenylic acids is formed and accumulated in the medium or in the cell bodies.

After the completion of the culture,.the cell bodies are removed, and inosinic acidand'adenylic acids are recovered from the fermentation liquor by an ion-exchange resin treatment, such. as that of Example 1, or by other known ion-exchange resin treatments, adsorption methods, percipitation methods or extraction processes.

The present invention is described in more detail in the following examples, which are, however, set forth merely by way of illustration andnot by way of limitation. In fact, they can be modified in various ways, well known to those versed inthe art, without deviation from the spirit or the scope of the invention as mentioned in the specification and theappended claims.

Example 1 The media employed inrthe present example are as follows.

:make upto one liter with water; pH beingadjusted at 7.0

with N NaOH before the sterilization.

Medium B:

Glucose g 50 2 4 15 KHgPO, g 5.0 K2HPO4 -g-.. g Guanine i mg 5 Xanthine' mg 5 Biotin ng" l makeup to. one liter with water; .pH being adjusted at 7.4 before the sterilization: After the sterilization 'CaCO which has been separately sterilized, is added to the solution in amount'of 1'0 g.,per liter. A30 ml. of the media is dividedly added to a 250' mlf-Erlenmeyers flask, and used after sterilization.

Micrococcus glutamicus No. A-101, which is a Xanthine-guanine-biotin requiring mutant (ATCC No. 14620, deposited at American Type Culture Collection, on March 2, 1962), is inoculated in the medium A and cultured at 28 C. for 24 hours. Micrococcus. glutamicus No. A-101 is obtained from MZ'CITOCOCCHS glutamicus ATCC 13032 by ultraviolet-ray irradiation and isolated by a selection method. Then, a 3 ml. of the cultured medium is-inoculated to the medium B, and incubated for 72 hours. After that time, the accumulations of-1200 g/ml. of 5'- inosinic acid, andSSO and 820,,itg./mlfof 3'- and- Sf-adenylic acids, respectively, as well as accumulation of' L- urease, neutralization may be accomplished by the addition of urea. A culturing period of 2 to 6 days is ordinarilyrequired with bacteria and'yeasts, whereas 4 to 10' glutamic acid at the same time, are observed. In the cell bodies also, inosinic acid and adenylic acid are accumu lated. The cell bodies and insoluble CaCO are removed from the fermented liquor by filtration and resulting filtrate is adjusted'to pH 82 byaddition of barium hydroxide and then to pH 9.0*by addition of sodium hydroxide to remove phosphoric acid and other impurities. The supernatant liquid is passed through a column filled with a strongly basic anion exchange, for example, dimethyl ethanol benzyl ammonium OI-Ltype (Dowex 2 OH type) to adsorb inosinic acid and adenylic .acid. After. washing the resin with water the adsorped iriosinicacid and adenylic acidare .eluteldjwith' a mixed solution containing 'formic acid and ammonium'formate in O.5:N and0.25 N concentrations,respectively, and'each fraction containing 5' lnosinic acid,- 3-adenylic"acid, or 5' aden'ylic'acid, are

recovered. The fractions containing the same compound are combined together, and each of the combined fractions is added with'barium hydroxide to precipitate the impurities, followed byfiltration of the impurities. Concentration and cooling of the filtrates give crude crystalline barium salts of each acid. The yields are 1.5 g. of inosinic acid, 780 mg. of 3-adenylic acid, and 910 mg. of 5'-adenylic acid. The barium salt is treated with H 80, to produce barium sulfate which is filtered or centrifuged off. The obtained liquor is further concentrated and added with ethanol thereby to precipitate nucleotides in the free acid form. These barium salts can be converted to the sodium salts by treatment with Na 'SO The resulting products are proved to be sodium salts of 5'-inosinic acid, 3'-adcnylic acid and 5'-adenylic acid, from the data of their elemental analyses, the analyses of the contents of base, sugar, ribose, and phosphoric acid, the periodic acid oxidations, optical rotation, and the ultraviolet absorption spectra.

Example 2 V Micrococcas glatamicus No. A-645 (ATCC No. 14619, deposited at American Type Culture Collection, on March 2, 1 962), which is a hypoxanthine-biotin requiring mutant is used. Micrococcus glatamicus No.7 A-645 is obtained from Micrococcas glutamicas ATCC 13032 by ultraviolet-ray irradiation and isolated by a selection method. The-pH of the medium is kept at about 6.0 during the incubation by addition of a urea solution. The fermentation medium used has a composition of 3% (weight percent based on the volume of the medium) of glucose, 1% of (NH SO 0.2% of KH PO ,,0.2% of K HPO 0.3% of corn steep liquor, and 25 mg./l. of hypoxanthine. The pH is controlled to 7.2 before sterilization. Other conditions are same as in Example 1.

During the cultivation, pH is controlled in such a manner that, after hours, 0.2-0.5 ml. of a 10% aqueous urea solution is added at every hour totaling to 5.2 ml. of the solution. After 76 hours cultivation, 1.2 mg./rnl. of 5'- 1 inosinic acid, 300 ,ag./ml. of 3-adeny1ic acid, and 640 deposited at American Type Culture Collection, on March 2, 1962), which is a thymine-requiring mutant and is obtained from Escherichia coli K-12 (obtained from Dr. F. J. Rayan of Columbia University) by a HNO treatment and isolated by a selection method, and keeping the pH of the medium approximately at 6.0 by addition, if necessary, of potassium hydroxide, and other procedures of Example 1 are repeated. During the cultivation, after about 10 hours, 2-3 drops (OJ-0.15 ml.) of 1 N KOH solution is added about every 3 hours, totalling to 3.4 ml. to control the pH. After 76hours cultivation, 1.8 rug/ml. of 5'-inosinic acid, 220 ,agjml. of 3'-adenylic acid, and 780 ,ag/ml. of 5-adenylic acid are accumulated. The medium employed for the seed culture is the same as the medium A in Example 1, excepting that no biotin is added. The fermentation medium employed contains 5% (weight percent based on the volume of the medium) of maltose, 1.5% of (NH SO 0.1% of KH PO 0.1% of K' HPO 0.2% of corn steep liquor, 0.1% of peptone, and 20, mg./l. of thymine, which is adjusted to pH 7.4 and sterilized before use.

Example 4 Bacillus subtilis NolA-1625 (ATCC No. 14617, deposited at American Type Culture Collection on March 2, 1962), which is a xanthineguanine-isoleucine-requiring 3 mutant, is inoculated to a seed medium containing 2% (weight percent based on the volume of the medium) of glucose, 1%. of meat extract, 0.5% of yeast extract.

1.0% of peptone, and 0.3% of NaCl, and adjusted to pH 7.0 Bacillus sub'tllis No. A-1625 is obtained from Bacillus sabtilis Marburg strain (obtained from Dr. D. M.

Bonner of Yale University) by an ultraviolet ray irradiation and isolated by a selection method. The fermentation medium contains 5% of glucose, 0.1% of KH PO 0.1% of K HPO 0.03% of MgSO -7H O, 0.05% of NZ-amine, 0.05% of glycine, 0.02% of aspartic acid, 0.02% of glutamic acid, 0.01% of glutamine, 0.3% of fumaric acid 0.01% of formic acid, 0.0005% of p-aminobenzoic acid, 0.0002% of folic acid, 25 g/ml. of guanine, 20 ,ag./ml. of xanthine, and 3 g./ml. of isoleucine, which is adjusted to pH 7.4 and sterilized before use. After 76 hours, cultivation as in Example 1, 2.2 mg./rnl. of 5- inosinic acid, 320 ig/ml. o-f 3-adenylic acid, and 965 gjrnl. of 5-adenylic acid are accumulated. If glycine, aspartic acid, glutamine, fumaric acid, formic acid, parninobenzoic acid, biotin, and folic acid are not added to the fermentation medium, the accumulations of inosinic acid and adenylic acid are reduced to a half amount.

7 Example 5 Using Bacillus sabtilis No. A-1203 (ATCC No. 14618, deposited at American Type Culture Collection on March 2, 1962), which is a hypoxanthine-thymine-isoleucine-requiring mutant, a cultivation is the same as in Example 4. The Bacillus subtilis No. A-946 is obtained from Bacillus subtilis Marburg strain (obtained from Dr. D. M. Bonner of Yale University) by an ultraviolet ray irradiation and isolated by a selection method. Fermentation medium used is the same as in Example 4 except that xanthine and guanine are replaced by 20 g/ml. of hypoxanthine and 20 ugJml. of thymine. After 82 hours cultivation, 1.6 mg./m1. of 5'-inosinic acid, 600 ,ag/ml. of 3-adenylic acid, and 1.0 mg./ml. of 5-adenylicacid are accumulated. V

7 Example 6 Using Aspergillas niger No. A-9'46, which is a guanineisoleuciue-requiring mutant, a cultivation is carried out, meanwhile the pH of the culture liquor is kept 6.2, by adding a urea solution timely and the other conditions are the same as in Example 4 except that, in the fermentation medium, xanthine is omitted. Aspergillas niger No. A-946 is obtained from Aspergillas nigerNRRL 337 by a C0 -ray irradiation and isolated by a selection method. After 10 hours from beginning of the cultivation, each' of 0.20.5 m1. of a 1 N NaON solution and a 10% aqueous urea solution are added at every 4-5 hours, totalling respectively to 3.6 ml. and 4.8 ml., to control the pH. After 6 days, 360 ag/ml, of 5-ino'sinic acid, 230 ag./ml.-of 3-adenylic acid, and 260 pg/ml. of 5-adenylic acid are accumulated.

Example 7 hours, a 10% urea solution and 1 N NaOH solution are added in respective amounts of 0.3-0.5 ml. and 2-3 drops (0.l-0.15 ml.) at every 4-7 hours, totalling to 4.7 ml. and-4.6 ml., respectively,to control the pH. After 6 days cultivation; 360 gJml. of 5?-inosinic acid,

'230 lg/ml. of 3- adenylic acid, and 260 ig/ml. of '5-adenylic acid are accumulated. a V 7 Example 8 Micrococeus glutamicas No. A-645 is used. The medium is made of glucose 20g, peptone 10 gr. yeast extract 2 g., sodium chloride 2g. and biotin 10 ag./l.'

and made up 1 l. with water. 10 ml. each of the medium (pl-I 7) is poured into test tubes and sterilized at'120 arsasee C; for. 10 min. The'micro-organism is inoculated to the medium; shaking culturedtat 28 C. for. 24 hours; and the cultureis used as a.seed* in-the. followingfermentation: The fermentation medium employed contains glucose 50g. KH PO 101g.,.K HPO 10g (NHQgSO 10 g.,.MgSO' =-7H O 0.3-g.,.p'eptone: 0.1 g.-', yeast extract 3v g;,.corn. steep liquor 1' g., and sodiumcitrate 0.5 g. inion'e literofwater', the pH being adjusted to 7.2. To 30: ml. each of. another. medium poured into 300 ml. Erlenmyer flasksand sterilized at:120 C. for 10 minutes; are added. 0.6 g..of CaCO reach. The seed culture is inoculatedi to the fermentation medium at the rate of 10% by volume. and clutured for 72 hours, with the result that. 3.8: mg./ ml. of '-inosinic. acid is formed.

Example 9 Example 8 is repeated with Micrococcas'glutamicus No. A-645, with the result of forming 4.6 mg./ml. of 5-inosinic acid and 1.2"mg./ml. of S-adenylic acid.

Example 10' Example 8 is repeated using Bacillus subtilis No. A4625, using'the result of formation of 4.5-mg./-ml. of 5'-inosinic acid andG.8.mg./ ml. of 3'-adenylic acid.

Example 11 Example 8- is repeated using Escherichia coli No. A-981, with the result of formation of 2.5 mg./ml. 5'-inosinic acid.

Examples 12-22 The parent strains are clutured as in the same Ways mentioned in respective foregoing examples. Neither inosinic acid nor adenylic acid is produced.

What We claim is:

1. A fermentation method which comprises (a). aerobically culturing Micrococcus glutamicus (ATCC 14620) under fermentation conditions in a fermentation mediurncontaining carbon source, nitrogensource, biotin and from-2 to 90 micrograms per milliliter each of Xanthine and guanine; whereby atleast one substance selected from the group consisting of 5'-inosim'c acid, 3'- adenylic acid land 5'-adenylic acid, is-directly produced and accumulated; (b) isolatingtheproduced-andaccumulated substance in aform selected from the groupconsi-sting of the-free acid and a salt thereof; and (c) recovering from said medium each-substance so produced and accumulated;

2. A'fermentation-method which comprises (a) aerobically culturingMicroc-occas glutamicas (ATCC 14619) under fermentation conditions in a fermentation medium containing carbon source, nitrogen source, biotin and from 2 to microgramsv per milliliter of hypoxanthine, whereby at least one substance selected fromthe group consisting of 5-inosinic acid; 3"-adenylic acid and 5- adenylic acidisdirectly produced and accumulated; (b) isolating the produced' and' accumulated substance in a form selected frorn the: group consisting of thefree acid and a salt thereof; (c) recovering from 'saidmedium each substance so produced'and accumulated.

3; A fermentation method which'comprises (a) aerobically culturing-Escherichia coli (ATGC 14621) under fermentation conditions in a' fermentation medium containing carbon source, nitrogen source and'from 2 to 90 micrograms per milliliter of thymine; whereby at least one substance selected from-the group-consisting of 5'- inosinic acid,- 3 -adenylic acid and 5'-adenylic acid is directly produced and accumulated; ('b) isolating theproduced-andaccumulatedsubstance in'a form selected from the group consisting of the free acid and a salt thereof; and (c) recovering from said medium each substance so producedand accumulated.

4; A fermentation method which comprises (a) aerobically'culturing. Bacillus subtilis' (ATCC 14617) under fermentation conditions in a fermentation medium. containing carbon. source, nitrogen source, isoleucine and from 2 to 90'micrograms per milliliter each of xanthine and guanine, whereby at least one substance selected from the group consisting of 5-inosinic acid; 3"-adcnylic' acid and 5'-adenylic acid is directly produced and accumtu lated; ('b) isolating the produced and accumulated substance in' a' form selected from the group consisting of'the freeiacid and a salt thereof; and (c)'recoveringfrom said medium'each substance so producedand accumulated.

5; A'fermentation method which comprises (a) aerobically culturingiBacillas'subtilis' (ATCC 14618) under fermentation conditions in a fermentation'medium con taining carbon' source, nitrogen source, isoleucine and from 2'-to* 90 micrograms per milliliter each' of hypoxanthine and thymine, whereby at least one substance se lected from the group" consisting of: 5'-inosinic acid, 3'-' adenylic acid and 5'-adenylic acid'is-directly'produced and accumulated; (b) isolating the produced and accumulated substance ina form selected from the group con sisting of the free acid anda salt thereof; and (c) recovering from said medium each substance so produced and accumulated.

References" Cited in the file of'this patent Journal of Biological Chemistry 235, 1474-1478 (I), 2103-2108, (II), 2672-2681 (III) (1960),.QP501J7. 

1. A FERMENTATION METHOD WHICH COMPRISES (A) AEROBICALLY CULTURING MICROCOCCUS GLUTAMICUS (ATCC 14620) UNDER FERMENTATION CONDITIONS IN A FERMENTATION MEDIUM CONTAINING CARBON SOURCE, NITROGEN SOURCE, BIOTIN AND FROM 2 TO 90 MICROGRAMS PER MILILITER EACH OF XANTHINE AND GUANINE, WHEREBY AT LEAST ONE SUBSTANCE SELECTED FROM THE GROUP CONSISTING OF 5''-INOSINIC ACID, 3''ADENYLIC ACID AND 5''-ADENYLIC ACID, IS DIRECTLY PRODUCED AND ACCUMULATED; (B) ISOLATING THE PRODUCED AND ACCUMULATED SUBSTANCE IN A FORM SELECTED FROM THE GROUP CONSISTING OF THE FREE ACID AND A SALT THEREOF; AND (C) RECOVERING FROMSAID MEDIUM EACH SUBSTANCE SO PRODUCED AND ACCUMULATED. 